The prolactin (PRL) gene regulates the egg production and incubation in laying chickens. Local chickens' reproductive systems will disrupt as a result of the incubation period activity, and they will lay fewer eggs. This study aimed to determine the prolactin gene polymorphism in IPB-D1 hens and its relationship to egg production. The polymorphism of the exon 5 prolactin gene was examined on 112 samples of the IPB-D1 chicken DNA collection from the Division of Animal Genetics and Breeding, Faculty of Animal Sciences, IPB University. By performing the phenol-chloroform method, the genomic DNA was obtained. A polymerase chain reaction (PCR) product with a size of 557 bp was produced as a result of the DNA amplification. Three single-nucleotide sequences were discovered. Three single-nucleotide polymorphisms (SNPs), g.7835A > G, g.7886A > T, and g.8052T > C, were found in exon 5 of the PRL gene. Each mutation was polymorphic and in Hardy-Weinberg equilibrium. The point mutation g.8052T > C significantly impacted the egg production of IPB-D1 chickens, according to the SNP association analysis on egg production, and may serve as a marker to enhance the selection for the features of egg production in IPB-D1 chickens.